This protocol is designed for extracting RNA from adherent cells growing on a plastic dish, or from a cell pellet. Normally it works best if you harvest directly from the plate.
- Prepare work space by cleaning thoroughly and by spraying with RNAse Away. Use pipettes and plastics that have been cleaned and only handled with gloved hands.
- Aspirate media from plated cells
- Wash gently 1 or 2 times with PBS, remove all PBS
- Add Trizol directly to the plate and mix extensively by pipetting all over the plate.
- 100 mm plates = 1 ml Trizol (independent of confluence)
- 1 well of a 6 well plate or a 35 mm plate = 500 µl Trizol
- 1 well of a 24 well plate or a 15 mm plate = 200 µl Trizol
- 1 thawed cell pellet from a 10 cm plate = 1 ml Trizol
- Transfer Trizol to 1.5 ml microfuge tube (mix well on plate, very goopy)
- Optional: Can add glycogen to the sample which may improve yield. It will remain with RNA as it is soluble in water. Do this if you have tiny amounts of starting material, otherwise omit this step.
- Incubate 5 minutes RT
- Optional: If there is a lot of insoluble material after a 5 minute RT incubation, can remove by centrifugation at 12,000 g for 10 min at 4º C before adding chloroform (a clear supernatant and jelly-like pellet should be seen). Transfer supernatant to a clean microfuge tube and proceed to next step. Only do this if you only want RNA, as it will destroy the DNA. There is an alternate procedure if you are also collecting DNA (see Trizol protocol from manufacturer)
- Add 200 µl chloroform (no isoamyl alcohol is needed) for every 1 ml of TRIzol used
- Shake vigorously for 15 seconds and incubate at room temp for 2-3 min.
- Centrifuge samples 15 min at 12,000 g at 4º C. After centrifugation you should see a clear layer on top, a red layer on bottom, and some solid white material between the two layers. If there is not good separation between the phases, spin again.
- Transfer upper (aqueous) phase to a clean tube. Save the lower phase for later DNA and/or protein extraction
- Add 0.5 ml of isopropanol per ml of TRIzol.
- Incubate at room temp for 10 min (if cloudy, precipitate for an additional 10 – 15 min).
- Centrifuge 12,000 g 10 min at 4ºC. After centrifugation a small white RNA should be visible on side of tube at this point. Can store as a pellet in the isopropoanol at 4ºC overnight at this point. Do not store at -20ºC and don’t go longer than overnight.
- Remove supernatant. Wash pellet by gently pipetting with 1 ml 75% ethanol for every 1 ml of TRIzol used. Mix by flicking and inverting tube or vortexing
- Centrifuge at 7,500 g 5 min at 4º C. Can store in 75% ethanol for 1 week at 4ºC or 1 year at -20ºC.
- Remove ethanol and air dry (preferable to vacuum drying). Pellet should be white or clear jelly-like.
- Resuspend in RNase free H2O (or DEPC-treated H2O). In general redissolve RNA from 5e6 cells in 50 µl. If pellet is over-dried, you can incubate at 55ºC for 10-15 min with repeated pipetting to completely dissolve.
- If using RNA for RT-PCR, treat with Dnase (Either use DNase I protocol or Turbo DNase by Ambion protocol)
- Expected yields depend on cell type and tissue type, but can expect the following:
- from 1e6 cells = 1 to 15 µg
- from 5 to 10e6 cells = 100 to 150 µg
- 1e4 cells = 140 ng (using glycogen)
- 10 mg tissue = 20 to 77 µg
- Expected A260/A280 ratio: Dissolved in H2O = 1.78, dissolved in TE = 2.2. Note: If ratio is < 1.65 see detailed manual for improving.